Download this PDF file - Journal of Tropical Life Science

THE JOURNAL OF TROPICAL LIFE SCIENCE
OPEN
ACCESS Freely available online
VOL. 4, NO. 3, pp. 210-215, November, 2014
Evaluation of Antioxidant Activity of Some Selected Tropical Fruits
in South Kalimantan, Indonesia
Efrilia Tanjung1, Muhammad Hafidz MS2, Iskandar Thalib2, Eko Suhartono3*
1
Datuk Sanggul General Hospital, South Kalimantan, Indonesia
Research Unit Mutiara Bunda Mother and Child Hospital, South Kalimantan, Indonesia
3
Medical Chemistry and Biochemistry Department, School of Medicine, Lambung Mangkurat of University,
South Kalimantan, Indonesia
2
ABSTRACT
In the present study antioxidant and antioxidant activity of some tropical fruits was evaluated. Antioxidants are
compounds or molecules that can scavenging and prevent free radicals and reactive oxygen species that can caused
a cell damage. Fruit was known as a source of antioxidant. South Kalimantan Indonesia, has a variety of fruit such
as mentega, nangka, timun suri and kuranji. Study for evaluating the antioxidant levels and activity of those fruit
were never been investigated. Thus, our study aim to measure the antioxidant levels and antioxidant activtity of
those selected fruits. Ascorbic acid, lycopene, b-carotene levels and antioxidant activity of four selected tropical
fruits was evaluated using spectrophotometer. The result of this studied suggest that the four selected tropical
fruits is potential antioxidant because it contained ascorbic acid, b-carotene, lycopene and had effect of scavenging
radical hydroxyl, hydrogen peroxide and chellating ferrous iron.
Keywords: antioxidant activity, ascorbic acid, b-carotene, lycopene
INTRODUCTION
Fruits are source of antioxidants that are beneficial
to health because it contains ascorbic acid, b-carotene,
lycopene, and others that can scavenging the free radical [1, 2]. Free radicals are atoms or molecules containing unpaired electrons therefore unstable molecule. It
can react with the cell membrane biomolecules such as
lipids, proteins, and carbohydrates. Because of that, the
presence of antioxidants is necessary to prevent or delay the damage [3, 4].
Antioxidants are compounds or molecules that can
scavenging and prevent free radicals and reactive oxygen species generated from the metabolism [4]. Plants
and animals maintain complex systems of multiple
types of antioxidants, such as glutathione, vitamin C,
vitamin A, and vitamin E as well as enzymes such as
peroxydase, catalase, superoxide dismutase and others.
Insufficient levels of antioxidants, or inhibition of the
antioxidant enzymes, cause oxidative stress and may
damage or kill cells [5].
*Corresponding author:
Eko Suhartono
Faculty of Medicine, University of Lambung Mangkurat,
Banjarmasin, Indonesia
E-mail: [email protected]
JTLS | J. Trop. Life. Science
In South Kalimantan Indonesia is a tropical country that has a variety of fruits that are consumed for
food and health [6]. For example methanol exctract
timun suri (Cucumis sativus) can be useful as an antiinflammatory, analgesic, and antioxidants [7, 8].
Nangka (Artocarpus heterolphyllus) can be useful as
anti-bacterial, anti-diabetic, anti-inflammatory, antioxidant and anti-helmintics [9]. Mentega (Diospyros
blancoi) can be useful as anti-diarrhea and antioxidant
[10].
The antioxidant properties mechanism of fruits
through the inhibiting initiation and breaking chain
propagation or suppressing formation of ROS by binding to the metal ions, reducing hydrogen peroxide, and
quenching superoxide and singlet oxygen [5]. The antioxidant activity of those fruits have not been investigated, therefore, many study should be performed. In
this present study, we investigate ascorbic acid, bcarotene, lycopene and antioxidant activity of kuranji,
mentega, timun suri, and nangka.
MATERIALS AND METHODS
Chemical and Materials
1% metaphosphoric acid, 2,6–dichloro phenolindophenol, acetone–hexane, 1mM FeCl3, 2 mM FeCl2,
210
Volume 4 | Number 3 | November| 2014
Evaluation of Antioxidant Activity of Some Selected Tropical Fruits
Hydroxyl Radical Scavenging Activity
The scavenging activity for hydroxyl radicals was
measured with Fenton reaction [12]. The absorbance of
the mixture at 560 nm was measured with a
Fruit Materials
spectrophotometer. Hydroxyl radical scavenging
Four types of tropical fruits and used as reference activity was calculated using the equation: (1were studied. They were kuranji (Dialium indum L), absorbance of sample/ absorbance of control) × 100.
mentega (Diospyros blancoi), timun suri (Cucumis Each experiment was carried out in triplicate and
sativus), and nangka (Artrocarpus heterolphyllus).
results averaged expressed as mean ± SD.
1mM ortophenanthroline, 0.2 M phosphate buffer (pH
7.8), 0.17 M H2O2, NaNO2, and AlCl3 were from
Sigma.
Ascorbic Acid Content
Ascorbic acid was determined according to the
method of Klein and Perry (1982). The dried
methanolic extract (100 mg) was extracted with 10 ml
of 1% metaphosphoric acid for 45 minute at room
temperature and filtered through whatman no. 4 filter
paper. The filtrate (1 ml) was mixed with 9 ml of 2,6dichlorophenolindophenol and the absorbance was
measured within 30 min at 515 nm against a blank.
Content of ascorbic acid was calculated on the basis of
the calibration curve of authentic L-ascorbic acid (0.020
–0.12 mg/ml). The assays were carried out in triplicate;
the results were mean values ± standard deviations and
expressed as mg of ascorbic acid/g of extract [11].
Chellating Effect of Ferrous Iron
The chelating effect of ferrous ions was estimated
by the method of Hung-Ju Chou et al. [13]. The
absorbance of the mixture was measured at 562 nm.
Chelating effect was calculated using the equation: (1 absorbance of sample/ absorbance of control) × 100.
Each experiment was carried out in triplicate and
results averaged expressed as mean ± SD.
Hydrogen Peroxide Scavenging Activity
The hydrogen peroxide scavenging was determined
according to the method of Ruch et al. [14]. The
absorbance value of the reaction mixture was recorded
at 230 nm. Hydrogen peroxide scavenging activity was
calculated using the equation: (1 - absorbance of
b-Carotene and Lycopene Content
sample/ absorbance of control) × 100. Each experiment
b-Carotene and lycopene were determined was carried out in triplicateand results averaged
according to the method of Nagata and Yamashita expressed as mean ± SD.
(1992). The dried methanolic extract (100 mg) was
vigorously shaken with 10 ml of acetone–hexane RESULTS AND DISCUSSION
mixture (4:6) for 1 min and filtered through whatman Ascorbic Acid
no. 4 filter paper. The absorbance of the filtrate was
Ascorbic acid is known as vitamin C is a substance
measured at 453, 505, 645 and 663 nm. Contents of b- commonly found in fruits [13-14]. Some studies
carotene and lycopene were calculated according to the suggest that the consumption of fruits and vegetables
following equations: lycopene (mg/100 ml) = -0.0458 are associated with reduced risks of diseases [15].
A663 + 0.372 A505-0.0806 A453 ; b-carotene (mg/100 Elderly people who take vitamin C and vitamin E
ml) = 0.216 A663- 0.304 A505+ 0.452 A453. The assays supplements have a 50% lower risk of dying
were carried out in triplicate; the results were mean prematurely from disease than do people who do not
values ± standard deviations and expressed as mg of supplement A Californian study has concluded people
carotenoid/g of extract [11].
who consume more than 750 mg/d of vitamin C
Table 1. Ascorbic Acid, b-Carotene and Lycopene of Four Selected Fruits
JTLS | J. Trop. Life. Science
211
Volume 4 | Number 3 | November | 2014
Efrilia Tanjung et al., 2014
reduce their risk of dying prematurely by 60% [15].
The result of ascorbic acid levels is shown in table
1. All four selected fruits contains ascorbic acid, and
mentega had a highest levels of ascorbic acid followed
by jackfruit, timun suri and kuranji. The levels of
ascorbic acid showed that the four selected fruits has a
potential antioxidant activity.
Ascorbic Acid is a water-soluble antioxidant. It was
first isolated in 1928, by the Hungarian biochemist and
nobel prize winner Szent-GyorGyi. It is an unstable,
easily oxidized acid and can be destroyed by oxygen,
alkali and high temperature [16].
Ascorbic acid has been shown to play important
role in several physiological processes in plants and
fruits, including growth, differentiation, and
metabolism [17]. Body requires ascorbic acid for
normal physiological functions. It helps in the
metabolism of tyrosine, folic acid and tryptophan. It
helps to lower blood cholesterol and contributes to the
synthesis of the amino acids carnitine and
catecholamine that regulate nervous system. It is
needed for tissue growth and wound healing. It helps
in the formation of neurotransmitters and increases the
absorption of iron in the gut. Being an antioxidant, it
protects the body from the harmful effects of free
radicals and pollutants [16].
Since ascorbic acid is water soluble, it can work
both inside and outside the cells to combat free radical
damages. Free radicals seek out an electron pair to
regain their stability. Ascorbic acid is an excellent
source of electrons therefore it can donate electrons to
free that radicals such as hydroxyl and superoxide
radicals and quench their reactivity [16].
Ascorbic acid prevents free radical damage in the
lungs and may even help to protect the central nervous
High doses given after but not before the injury
successfully suppressed preventing edema [16].
b- Carotene and Lycopene
Carotenoids, such as lycopene and b-carotene, are
natural constituents of many plants and may protect
against disease. Carotenoids are a family of pigmented
compounds that are synthesized by plants and
microorganisms but not animals. In plants, they
contribute to the photosynthetic machinery and protect
them against photo-damage [18].
Fruits and vegetables constitute the major sources
of carotenoid in human diet. They are present as
micro-components in fruits and vegetables and are
responsible for their yellow, orange and red colors.
Carotenoids are thought to be responsible for the
beneficial properties of fruits and vegetables in
preventing human diseases including cardiovascular
diseases, cancer and other chronic diseases [18].
In recent years the antioxidant properties of
carotenoids has been the major focus of research. More
than 600 carotenoids have so far been identified in
nature. However, only about 40 are present in a typical
human diet. Of these 40 about 20 carotenoids have
been identified in human blood and tissues. Close to
90% of the carotenoids in the diet and human body is
represented by b-carotene, α-carotene, lycopene, lutein
and cryptoxanthin [18].
The levels of b-carotene and lycopene in four
selected fruits shown in table 1. The levels of bcarotene decreased in the order of mentega fruit >
jackfruit > kuranji > timun suri, and the levels of
lycopene decreased in the order of mentega fruit >
jackfruit > kuranji > timun suri.
b-carotene is commonly known as a radical
Table 2. Antioxidant Activity of Four Selected Fruits
system problems from such damage. In a study of
guinea pigs, pretreatment of ascorbic acid including
effectively diminished the acute lung damage caused by
the introduction of super oxide anion free oxygen
radicals to the trachea. Ascorbic acid also has been
tested as an antioxidant inflammatory reaction in mice.
JTLS | J. Trop. Life. Science
scavenger and a physical scavenger of singlet oxygen
and is believed to play an important role in the
inhibition of initial stages of lipid peroxidation [21].
The antioxidant properties of b-carotene have been
suggested as being the main mechanism by which they
afford their beneficial effects. Recent studies are also
212
Volume 4 | Number 3 | November | 2014
Evaluation of Antioxidant Activity of Some Selected Tropical Fruits
showing that carotenoids may mediate their effects via
other mechanisms such as gap junction
communication, cell growth regulation, modulating
gene expression, immune response and as modulators
of phase I and II drug metabolizing enzymes.
However, carotenoids such as α- and b-carotene and bcryptoxanthin have the added advantage of being able
to be converted to Vitamin A and its related role in the
development and disease prevention [18].
Several in vitro, animal and human experiments
have demonstrated the antioxidant properties of
carotenoids such as b-carotene. It is interesting to
observe that b-carotene has also been reported to act as
a pro-oxidant under certain situations. b-carotene at a
concentration of 0.2 mM augmented UVA-induced
haem oxygenase-1 induction indicating a pro-oxidant
effect. The pro-oxidant effect of b-carotene was also
demonstrated in rats that showed increased activity of
phase I enzymes in liver, kidney and intestine as well
as increased oxidative stress [18].
Lycopene is present in many fruits and vegetables,
with tomatoes and processed tomato products being
among the richest sources. Several recent studies
suggest that dietary lycopene is able to reduce the risk
of chronic diseases such as cancer and cardiovascular
diseases. Although several mechanisms have been
implicated in health-beneficial effects of lycopene, such
as modulation of intercellular gap junction
communication, hormones, immune system and
metabolic pathways, the antioxidant properties of
lycopene are thought to be primarily involved in its
preventive effects in chronic diseases. Because of its
high number of conjugated dienes, lycopene is one of
the most potent antioxidants, with a singlet-oxygenquenching ability twice as high as that of β-carotene
and 10 times higher than that of α-tocopherol [21].
Hydroxyl Radical Scavenging Activity
Unlike superoxide, which can be detoxified by
superoxide dismutase, the hydroxyl radical cannot be
eliminated by an enzymatic reaction. Mechanisms for
scavenging peroxyl radicals for the protection of
cellular structures includes dietary antioxidants such as
flavonoid and vitamin C [20, 21].
Radical hydroxly is the most reactive among all
ROS. It has a single unpaired electron, thus, it can
react with oxygen in triplet ground state. Radical
hydroxyl interacts with all biological molecules and
causes subsequent cellular damages such as lipid
peroxidation, protein damage, and membrane
destruction. Because cells have no enzymatic
JTLS | J. Trop. Life. Science
mechanism to eliminate radical hydroxyl, its excess
production can eventually lead to cell death. The
oxidation of organic substrates by radical hydroxyl may
proceed by two possible reactions, either by addition of
radical hyroxyl to organic molecules or due to
abstraction of a hydrogen atom from it [17].
The hydroxyl radical scavenging activity of the
various extracts was investigated in this study (table 2).
All extracts almost have equal ability to scavenging the
hydroxyl radical. Kuranji has the highest abilities to
scavenging hydroxl radical than mentega fruit, timun
suri and jackfruit.
Chellating Effect of Ferrous Iron
The ferrous ion chelating activities of mentega fruit,
kuranji, pasak timun suri, and jackfruit extracts are
shown in table 2. The metal scavenging effect of these
samples decreased in the order of mentega fruit >
kuranji > timun suri > jackfruit.
Although iron is vital for life, it can be toxic when
it is present in excess. Iron homeostasis is a complex
process, as there are many different proteins that
respond not only to the total body burden of iron, but
also to stimuli such as hypoxia, anemia and
inflammation [22].
About 65% of iron is bound to hemoglobin, 10% is
a constituent of myoglobin, cytochromes, and ironcontaining enzymes, and 25% is bound to the iron
storage proteins, ferritin and hemosiderin. About 0.1%
of body iron circulates in the plasma as an
exchangeable pool, essentially all bound to transferrin.
The process of chelation not only facilitates the
transport of iron into cells, but also prevents ironmediated free radical toxicity [22].
The iron-mediated free radical toxicity leading to
the formation of hydroxyl radicals and hydroperoxide
decomposition reactions via Fenton reaction [23]. Fe2+
caused the production of oxyradicals and lipid
peroxidation, therefore the ability of substances to
chelating iron can be use as a valuable antioxidant. It
was reported that the presence of chelating metal such
as iron is capable of generating free radicals from
peroxides and may be implicated in human
cardiovascular disease [1, 23, 24].
Many studies documented that mutations in
superoxide dismutase enzymes and iron-uptake
regulator may lead to excess levels of superoxide anion
radicals and iron overload. Such a condition leads to
the possibility of redox active iron to participate in
organic and inorganic oxygen radical reactions, such as
stimulating lipid peroxidation and catalyzing the
213
Volume 4 | Number 3 | November | 2014
Efrilia Tanjung et al., 2014
formation of damaging hydroxyl radicals with
subsequent tissue damage [22].
Hydrogen Peroxide Scavenging Activity
Hydrogen peroxide itself is not very reactive, but it
can sometimes be toxic to cell because of it may
increase the hydroxyl radical in the cells.
In mammalian cells, potential enzymatic sources of
ROS include the mitochondrial electron transport
chain, the arachidonic acid metabolizing enzymes
lipoxygenase and cycloxygenase, the cytochrome
P450s, xanthine oxidase, NADPH oxidases, uncoupled
nitric oxide synthase, peroxidases, and other
hemoproteins. These systems primarily catalyze one
electron reduction of molecular oxygen to form radical
superoxide which rapidly inactivates NO• to form
peroxynitrite. Under ambient conditions, some radical
superoxide is dismutated to hydrogen peroxide
spontaneously or catalyzed by superoxide dismutase.
Of interest, loss of NO• could lead to enhanced
formation of hydrogen peroxide. Some enzymes, such
as xanthine oxidase and glucose oxidase, can directly
produce hydrogen peroxide by donating two electrons
to oxygen. In the presence of heavy metals, hydrogen
peroxide undergoes Fenton reaction to form highly
reactive hydroxyl radical [24].
Thus removing H2O2 is very important throughout
food systems. The composition of hydrogen peroxide
into water may occur according to the antioxidant
compounds as the antioxidant component present in
the extract are good electron donors, they may
accelerate the conversion of H2O2 to H2O [25-27].
The hydrogen peroxide scavenging activities of of
mentega fruit, kuranji, pasak timun suri, and jackfruit
extracts are shown in table 2.
Table 2 showed that the four selected fruits have a
potential hydrogen peroxide scavenging activity.
Timun suri has a higher scavenging activity and
followed by mentega fruit, kuranji and jackfruit.
highest hydroxyl radical scavenging activity and
followed by mentega fruit, timun suri and jackfruit. For
hydrogen peroxide scavenging activity, timun suri have
a highest activity than mentega fruit, kuranji and
jackfruit.
REFERENCES
CONCLUSIONS
In present study the ascorbic acid, b-carotene,
lycopene and antioxidant activity of four selected
tropical fruits was evaluated. The levels of ascorbic acid
decerased in the order of mentega fruit > jackfruit >
timun suri > kuranji. The levels of b-carotene and
lycopene decreased in the order of mentega fruit >
jackfruit > kuranji > timun suri. Mentega fruit have a
highest chellating effect on ferrous iron followed by
kuranji, timun suri and jackfruit. Kuranji have a
JTLS | J. Trop. Life. Science
214
1.
Suhartono E, Viani E, Rahmadhan MA, Gultom IS,
Rakhman MF, Indrawardhana D (2012) Total flavonoid
and antioxidant activity of some selected medicinal plants
in South Kalimantan of Indonesian. APCBEE Procedia. 4:
235-239.
2. Karadeniz F, Burdurlu HS, Koca N, Soyer Y (2005)
Antioxidant activity of selected fruits and vegetables
grown in Turkey. Turk J Agric For. 29: 297-303.
3. Nadeesha MKF, Bamunuarachchi, Edirisinghe EMRKBM,
Weerasinghe WMSK (2007) Studies on antioxidant
activity of Indian gooseberry and seed. J. Sci. Univ.
Kelaniya. 3: 83-92.
4. Aris SRS, Mustafa S, Ahmat N, Jaafar FM (2009) Phenolic
content and antioxidant activity of fruits of Ficus
deltiodea var Angustifolia Sp. MJAS. 13: 146-150.
5. Lim YY, Lim TT, Tee JJ (2007) Antioxidant properties of
several tropical fruits : A comparative study. Food
Chemistry. 103: 1003-1008.
6. Soeksmanto A, Hapsari Y, Simanjuntak P (2007)
Antioxidant content of parts Mahkota Dewa, Phaleria
macrocarpa (Scheff) Boerl (Thymelaceae). Biodiversitas. 8:
92-95.
7. Gill SN, Sood S, Muthuraman A, Garg M, Kumar R, Bali
M, Sharma PD (2010) Antioxidant, anti-inflammatory and
analgesic potential of Cucumis sativus seed extract. Lat.
Am. J. Pharm. 29: 927-932.
8. Agarwal M, Kumar A, Gupta R, Upadhyaya S (2012)
Exctraction of polyphenol, flavonoid from Embica
Officinalis, Citrus limon, Cucumis sativus and evaluation
of their antioxidant activity. Orient. J. Chem. 28: 993-998.
9. Shanmugapriya K, Saravana PS, Payal H, Mohammed P,
Binnie W (2011) Antioxidant activity, total phenolic and
flavonoid content of Artocarpus heterophyllus and
Manilkara zapota seeds and its reduction potential. Int J
Pharm Pharm Sci. 3: 256-260.
10. Howlader SI, Rahman M (2012) Antioxidant and
antidiarrhoeal Potentiality of Diosypros blancoi. Int. J.
Pharmacol. 8: 403-409.
11. Chou HJ, Kuo JT, Lin ES.(2009) Comparative antioxidant
properties of water extracts from different parts of
beefsteak plant (Perilla frutescens). JFDA. 17: 489-96.
12. Ruch RJ, Cheng SJ and , Klaunig JE. (1989) Prevention of
cytotoxicity and inhibition of intracellular communication
by antioxidant catechins isolated from Chinese green tea.
Volume 4 | Number 3 | November | 2014
Evaluation of Antioxidant Activity of Some Selected Tropical Fruits
Carcinogenesis. 10: 1003–1008.
13. Cioroi M. (2007) Study on L-Ascorbic Acid Contetnts
from Exotic Fruits. Cercetări Agronomice în Moldova
Anul XXXX. 1: 23-27.
14. Phillips KM, Tarrago MT, Gebhardt SE, Exler J, Patterson
KY, Haytowitz DB, Pehrsson PR, Holden JM (2010)
Stability of vitamin C in frozen raw fruit and vegetable
homogenates. Food. Comp. Anal. 23 : 253–259.
15. Rekha C, Poornima G, Manasa M, Abhipsa V, Devi JP,
Kumar HTV, Kekuda TRP (2012) Ascorbic acid, total
phenol content and antioxidant activity of fresh juices of
four ripe and unripe citrus fruits. Chem Sci Trans. 1: 303310.
16. Iqbal K, Alam K, M. Muzaffar A.K.K (2004) Biological
significance of ascorbic acid (vitamin C) in human health.
Pak J Nutrition. 3: 5-13.
17. Sharma P, Ambuj BJ, Rama SD, M. Pessarakli. (2012)
Reactive oxygen species, oxidative damage, and
antioxidative defense mechanism in plants under stressful
conditions. Journal of Botany. 1-26.
18. A.V. Rao, L.G. Rao. (2007) Carotenoids and human
health. Pharmacological Research. 55 : 207-216.
19. Paolo P, Assunta C, Rosella S, Achille C (2012) Lycopene
as a guardian of redox signalling. Acta Biochimica
Polonica. 59: 21-25.
20. Chiosa V, Mandravel C, Kleinjans JCS, Moonen E (2005)
Determination of β-carotene concentration in orange and
apple juice and in vitamin supplemented drinks. Chimie
Anul XIV (serie nouă). I-II : 253-258.
JTLS | J. Trop. Life. Science
215
21. Batool F, Sabir SM, Rocha JBT, Shah HA, Saify ZS,
Ahmed SD (2010) Evaluation of antioxidant and free
radical scavenging activities of fruit exctract from
Zanthoxyllum alatum : a commonly used spice from
pakistan. Pak. J. Bot. 42: 4299-4311.
22. K. Jornova, M. Valko. (2011) Advances in metal-induced
oxidative stress and human disease. Toxicology. 283: 6587.
23. Sudha G, Priya S, Shree RI, Vadivukkarasi S (2011) In
vitro free radical scavenging activity of raw pepino fruit
(Solanum muricatum aiton). Int J Curr Pharm Res. 3: 137
– 140.
24. Elekofehinti OO, Kade IJ (2012) Aqueous extract of
Solanum anguivi Lam. Fruits (African Egg Plant) inhibit
Fe2+ and SNP induced lipid peroxidation in Rat’s brain –
in vitro. Der Pharmacia Lettre. 4: 1352-1359.
25. Behyan O, Elmastas M, Gedikli F (2010) Total phenolic
compounds antioxidant capacity of leaf, dry fruit and
fresh fruit of feijoa (Acca sellowiana, Myrtaceae). J. Med.
Plant. Res. 4: 1065-1072.
26. Pal R, Girhepunje K, Shrivastav N, Hussain MH,
Thirumoorthy (2011) Antioxidant and free radical
scavenging activity of ethanolic extract of Morinda
citrifolia. Annals of Biological Research. 2: 127 – 131.
27. Suhartono E, Viani E, Rahmadhan MA, Gultom IS,
Rakhman MF, Indrawardhana D (2012) Screening of
medicinal plant for total flavonoid and antioxidant activity
in South Kalimantan of Indonesian. IJCEA. 3: 297-299.
Volume 4 | Number 3 | November | 2014